The strategies under Goals above involve several steps that together comprise a push-pull approach. First, we optimize the antigen to improve immunogenicity by epitope enhancement, increasing affinity for MHC. We have done this for 2 new prostate cancer antigens, TARP and POTE. We fully accrued a phase I/II TARP clinical trial in D0 prostate cancer patients with rising PSA levels using a TARP peptide that we epitope-enhanced to improve HLA-A2 binding and a second high affinity one we mapped. The slope of PSA rise significantly decreased among 72% of 40 patients (p = 0.0012) at 24 wks and 74% (p = 0.0004) at 48 wks, implying slowing of cancer growth. A randomized placebo-controlled phase II trial is now open and accruing patients. This has a 6--patient lead-in accrual to test safety of additional TARP peptides followed by a 2:1 randomization (44:22) of vaccine to placebo. The second step is to push the response with molecular adjuvants, such as cytokines, Toll-like receptor (TLR) ligands and NKT agonists, to improve not only the quantity but also the quality of the response. We published that IL-15 is a key mediator of CD4 T cell help for CD8 T cells and that IL-15 increased the CD8 T cell avidity, needed for effective clearance of virus or tumor cells. We translated this to humans showing that IL-15 could substitute for CD4 help to induce a primary in vitro CD8 T cell response of naive T cells, and restored alloresponsiveness of CD8 T cells from HIV-infected patients to normal levels. We also found that IL-1beta as adjuvant could enhance CD8 T cell responses and skew CD4 help to Th17. We found surprisingly that the Th17 CD4 cells were not good helpers for a CD8 T cell response as measured by IFNgamma production, but rather they skewed the CD8 response to IL-17 production through an effect on DCs dependent on IL-21 & 23. We also investigated TLR ligands as adjuvants to mature DCs and induce production of IL-12 and IL-15. We identified in mice a synergistic triple TLR ligand combination and tested this with IL-15, both or neither as vaccine adjuvants in a peptide-prime, MVA-boost mucosal vaccine for SIV in macaques, challenging intrarectally with SIVmac251. Only macaques receiving both showed partial protection. In the adaptive immune arm, only polyfunctional CD8 T cells specific for SIV antigens correlated with protection. In the innate immune arm, the adjuvants induced long-lived innate protection by APOBEC3G. These adjuvants also increased CD4 cell preservation in the gut, independent of viral load. Adding a PD-1 blocker and an NKT cell agonist led to substantial CD8-dependent protection (after intrarectal SIV challenge) induced by adjuvant alone. Yet vaccine could induce MDSC counteracting vaccine efficacy. The third step is to target the immune response to the relevant tissue, the mucosa in the case of HIV. We published a novel nanoparticle approach to vaccine delivery to the large intestine, using vaccine nanoparticles coated with Eudragit FS30D to allow oral delivery and release of the particles primarily in the large intestine, bypassing the stomach and small intestine. This effectively substituted for intrarectal delivery to protect against rectal or vaginal viral challenge. Moreover, the novel approach allows selective oral delivery to the small or large intestine, making it possible to distinguish immunization in these compartments for the first time. We have recently adapted this approach to non-human primates in an AIDS vaccine. 2/7 animals so immunized were protected from acquisition of SHIVsf162P4 high dose rectal challenge (p=0.04 vs 0/29 controls). An expanded study showed significant protection by an oral nanoparticle vaccine incorporating full-length single chain Env-CD4D1-D2 fusion protein (FLSC) and MVA against repeated low-dose intrarectal challenge. Among control animals from different sources, key determinants of susceptibility were colorectal inflammatory target cells and certain gut microbiota that affect these. If the small & large intestine are distinct compartments, homing of T cells must be different. We found that homing to the large intestine is governed by DCs from colon patches, using a mechanism involving alpha4beta7 but not CCR9, distinct from that in the small intestine. We are identifying chemokines to selectively target T cells to the colon. In contrast to previous belief, we discovered that CD103+ DCs could patrol the lumen of the colon in crypts associated with colon patches, attracted by CCL20, capture bacteria and bring them back to the lamina propria. We also published, contrary to accepted dogma, that the type 2 mucosa of the vagina can serve as an inductive site for priming of naive CD8 T cells without help from draining lymph nodes. The fourth step is to remove the brakes, i.e., block negative regulatory mechanisms that inhibit immunity. We previously discovered a new immunoregulatory pathway involving NKT cell suppression of tumor immunity, dependent on IL-13 and TGF-beta. We found that type I NKT cells (using an invariant TCRalpha chain) protected, whereas type II NKT cells (using diverse TCRs) suppressed immunity, and that these subsets cross-regulated each other, defining a new immunoregulatory axis that could influence subsequent adaptive immune responses. We published that two different regulatory cells (Tregs & type II NKT) suppressed immunity to the same tumor simultaneously but independently, and found that a third T cell, the type I NKT cell, can determine the balance between these, regulating the regulators. As humans with cancer often have a deficiency of type I NKT cell function, they may require blockade of both T regs and type II NKT cells to reveal tumor immunity. We also recently developed a way to make sulfatide-loaded CD1d multimers that can stain type II NKT cells, allowing detection of these otherwise elusive cells, and found that they are large granular lymphocytes arising independently of PLZF predominantly in the lung and liver, frequent sites of tumor metastases. Conversely, stimulating with a type I NKT cell agonist can protect against tumors. We discovered the first of a new class of NKT agonist, beta-mannosylceramide, that protects against cancer by a mechanism different from that of the classic a-GalCer, being dependent on TNF-alpha and nitric oxide synthase rather than on interferon-gamma. We have also found that it does not induce long-term anergy induced by a-GalCer. B-ManCer synergizes with a cancer vaccine in mice and also stimulates human NKT cells, suggesting translation to human cancer therapy. We are studying it in combination with a novel chemotherapy that induces immunogenic cell death and tumor immunity. A key mediator of the NKT regulatory pathway and an important regulator of T regulatory cells is TGFbeta. We found that blockade of TGFbeta can protect against certain tumors in mice, and can synergize with anti-cancer vaccines in 2 mouse models, dependent on CD8 T cells. We have translated this into a clinical trial of a human anti-TGFbeta monoclonal antibody in a CRADA with Genzyme, and published that in melanoma it is safe and has some anti-cancer activity as a single agent. We found that blocking TGFb1&2 is sufficient without TGFb3. Finally, we found that an adenovirus vaccine expressing the extracellular and transmembrane (ECTM) domains of HER-2 can cure large established mammary cancers and lung metastases in mice. The mechanism depends on antibodies that inhibit HER-2 function, and is FcR independent, unlike Herceptin. We made a similar cGMP recombinant adenovirus expressing the human HER-2 ECTM domains used to transduce autologous dendritic cells as a vaccine in a clinical trial in HER-2+ cancer patients, and already at the second dose level, 3/6 patients with advanced metastatic cancers had objective responses.